Microbial Growth

Bacterial Cell Division

Binary Fission (6.1)
- Growth is defined as an increase in the number of cells, representing population growth.
- Bacterial population dynamics involve reproduction, death, and the intermediate states.
- Example: Starting with 100 microorganisms and a generation time of 20 minutes under unlimited nutrient supply results in 72 reproduction cycles per day, leading to $4.72 \times 10^{23}$ cells. If this growth continues for a week, it reaches approximately 2 trillion tons, equivalent to the total global biomass.
Binary Fission Cycle
1. Chromosome replication begins.
2. Each chromosome attaches to the plasma membrane as the cell wall and plasma membrane start to divide.
3. DNA replication continues.
4. New cell walls and cell membranes completely divide the cell into two new cells, resulting in daughter cells separating.
Generation Time
- Generation time is the time required to complete a binary fission cycle.
- It varies depending on nutrition, environmental factors, and genetic factors.
- The cycle involves DNA replication, synthesis of cell division-specific proteins, duplication of cellular machinery, and synthesis of membrane and cell wall.

Fts Proteins (6.2)

Filamentous temperature-sensitive proteins are essential for cell division in all prokaryotes.
Temperature-sensitive mutants of Fts proteins tend to elongate into a filamentous form.
Fts proteins can be deactivated due to other environmental stresses, including nutrient limitation.
Divisome Formation
- Fts proteins interact to form a division apparatus in the cell called the divisome.
- Divisome forms after DNA replication.
- FtsZ: Forms a ring around the center of the cell.
  - Its location is facilitated by Min proteins.
  - Directs cell division at the central plane of the cell.
  - Involves GTP-dependent polymerization/depolymerization.
  - Acts as a marker versus having a contractile function.
- ZipA: An anchor that connects the FtsZ ring to the cytoplasmic membrane.
- FtsA: Helps connect the FtsZ ring to the membrane and also recruits other divisome proteins.

Cell Morphology Determinants (6.3)

Prokaryotic cells are not amorphous bags; they have a defined structure.
Recent findings (late 1990s - early 2000s) revealed the presence of a prokaryotic cytoskeleton.
Cytoskeleton: A dynamic scaffolding within cells, providing structural integrity, shape, and intracellular organization.
- Actin-like protein (FtsZ)
- Tubulin-like protein (MreB)
- Intermediate filament-like protein (Crescentin)
MreB
- Forms a simple cytoskeleton in cells of Bacteria and likely Archaea.
- Forms spiral-shaped bands around the inside of the cell, underneath the cytoplasmic membrane.
- Not found in coccus-shaped bacteria.
- Localizes synthesis of new peptidoglycan and other cell wall components.
Crescentin
- In vibrio (curved-rod) shaped bacteria.
- Localized onto the concave face of the curved cells.
Archaea have cell morphology determinants similar to bacterial MreB and FtsZ.

Peptidoglycan Synthesis (6.4)

A major event in cell division.
Three steps: Splicing, growth, and transpeptidation.
- Splicing
  - Small opening in the wall.
  - Autolysins cut $β$ -1,4 glycosidic bonds.
- Growth
  - Insertion of peptidoglycan precursors.
  - Glycosylases facilitate this.
  - Bactoprenol acts as a precursor carrier and interacts with glycosylases.
  - Wall band: The junction between new and old peptidoglycan.
- Transpeptidation
  - Final step in cell wall synthesis.
  - Forms the peptide cross-links between muramic acid residues.
  - Involves the removal of D-Ala which is exergonic, providing the driving force.
  - FtsI protein is the key protein.
  - Inhibited by the antibiotic penicillin.

Growth of Bacterial Populations

Exponential Growth (6.5)
- Growth is an increase in the number of cells.
- Generation time (doubling time) is the time required to produce two new cells or the time for the cell number to double.
- Varies greatly depending on type of organism, temperature, nutrients, and other conditions.
- Cell number increases exponentially.
Mathematics of Exponential Growth (6.6)
- Doubling follows a geometric progression of the number 2: $N = N_02^n$ , where:
  - $N$ : the final cell number
  - $N_0$ : the initial cell number
  - $n$ : the number of generations
- Generation time ( $g$ ) is calculated as $g = t/n$ , where:
  - $t$ : the duration of exponential growth
  - $n$ : the number of generations
Growth Cycle/Growth Phase in Batch Culture (6.7)
- Lag Phase: Preparatory period where cells produce cellular machinery for the new environment. The duration depends on the history of the inoculum.
- Exponential (Growth) Phase: Population doubles per generation; determined by nutrient availability and rate of end product accumulation.
- Stationary Phase: Growth rate equals zero (stopped growth); cryptic growth (growth equals death).
- Death Phase: Cell lysis exceeds cell growth; cells go into a dormant stage.

Measuring Microbial Growth

Total Cell Counts (6.9)
- Number of microorganisms per volume sample (often mL), e.g., $6.0 \times 10^8/mL$ .
- Dry Count: Involves fixing, staining, and counting.
- Wet Count: Requires a special counting chamber (Petroff-Hausser counting chamber).
  - The chamber (whole grid area) holds 0.02 mL sample.
- Advantages: Easy, fast sample preparation; reasonable estimation.
- Disadvantages: Not accurate; cannot distinguish dead cells; only good with a small concentration window (either concentrated or diluted); moving cells may be counted multiple times or missed; hard to distinguish debris.
Viable Cell Count (Plate Counts) (6.10)
- Counts only viable cells (living, reproducing cells).
- Better representation of samples in pathogenic microbiology, but not ideal for environmental samples.
- Disadvantages: Not accurate; cultivation is selective; only good with a small concentration window.
- Two main methods:
  - Spread-plate method
  - Pour-plate method
    Proper dilution is needed to obtain countable colonies.\
    Example plate count calculation: If a 1-mL sample from 1/10^3 dilution plate results in 159 colonies, then cell/ml $= 159 \times 10^3= 1.59 \times 10^5$ .
The Great Plate Count Anomaly
- Direct microscopic counts often exceed recoverable counts on plates.
- Reasons:
  *Different organisms in a sample have vastly diverse requirements for resources and conditions in lab culture.
  *Microscopic methods count dead cells whereas viable methods do not.
Turbidimetric Method (6.11)
- Turbidity measurements are an indirect but rapid method of measuring microbial growth.
- Most often measured with a spectrophotometer, referred to as optical density (O.D.).
- Works well at turbidity (Optical Density, OD) range between 0.1 and 1.0; may need dilution.
- Advantages: Non-destructive, fast, and reliable.
- Disadvantages: Can be problematic with biofilm or clumping; standard curve is needed first.

Temperature

Temperature is a major environmental factor controlling microbial growth.
Cardinal temperatures: Minimum, optimum, and maximum temperatures at which an organism grows.
- Minimum: Membrane gelling; transport processes so slow that growth cannot occur
- Optimum: Enzymatic reactions occurring at maximal possible rate
- Maximum: Protein denaturation; collapse of the cytoplasmic membrane; thermal lysis
Microorganisms classified by growth temperature optima:
- Psychrophile: Low temperature (optimum < $15^oC$ )
- Mesophile: Midrange temperature (optimum 20 – $40^oC$ )
- Thermophile: High temperature (optimum 45 – $60^oC$ )
- Hyperthermophile: Very high temperature (optimum > $80^oC$ )
Growth at Cold Temperatures (6.13)
- Psychrophilic:
  - Maximum: below $20^oC$
  - Minimum: below $0^oC$
  - Optimum: below $15^oC$
- Psychrotolerant:
  - Minimum: below $0^oC$
  - Optimum: 20 – $40^oC$
  - Most mesophiles are psychrotolerant.
Molecular Adaptations to Psychrophily
- Characteristics of cold-active enzymes:
  - More $α$ -helices than $β$ -sheets
  - More polar and less hydrophobic amino acids
  - Fewer weak bonds
  - Decreased interactions between protein domains
- Membrane structure/transport:
  - High unsaturated fatty acid contents that work best at low temperatures.
Growth at High Temperatures (6.14)
- Thermophile: Optimum: 45 - $60^oC$
- Hyperthermophile: Optimum: above $80^oC$
Molecular Adaptations to Thermophily
- Characteristics of hot-active enzymes:
  - Critical amino acid substitutions in a few locations lead to heat-tolerant folding.
  - Increased number of ionic bonds.
  - Production of solutes (e.g., di-inositol phosphate, diglycerol phosphate) help stabilize proteins.
- Membrane structure:
  - Bacteria have lipids rich in saturated fatty acids.
  - Archaea have a lipid monolayer rather than a bilayer.

Other Environmental Factors

Effects of pH (6.15)
- Neutrophile: pH 6-8 (most microorganisms)
- Acidophile: Below pH 6 (fungi are generally more acid-tolerant)
- Alkaliphile: Above pH 9 (some are also halophiles)
- Internal pH is maintained near neutral pH.
Osmotic Effects (6.16)
- Water activity ( $a_w$ ): Defined as ratio of vapor pressure of air in equilibrium with a substance or solution to the vapor pressure of pure water; reflects free water availability
- Halotolerant
- Halophile:
  - Mild halophile: 1-6%
  - Moderate halophile: 7-15%
  - Extreme halophile: 15 – 30%
- Xerophile: dry (many molds)
- Osmophile: high sugar (many yeasts)
- Compatible solutes: Increase internal solute concentration, non-inhibitory to cellular reactions; mainly sugars, sugar alcohols, and amino acids.
Oxygen (6.17-18)
- Aerobes: Require oxygen to live.
- Anaerobes: Do not require oxygen and may be killed by exposure to oxygen.
- Facultative organisms: Can live with or without oxygen (aerobic respiration + fermentation).
- Microaerophiles: Can use only low level oxygen.
- Aerotolerant anaerobes: Can tolerate oxygen and grow in oxygen even though they cannot use it (obligate fermenters).
Reactive Oxygen Species
- Produced by $O2$ reduction or $H2O$ oxidation.
 - Singlet oxygen ( $^1O_2$ ): Excited state of oxygen; often light-driven, short half-life; phototrophic microorganisms.
 - Superoxide ( $O_2^-$ ): Strong oxidant; damages all organic compounds.
 - Hydrogen peroxide ( $H2O2$ ): Less strong oxidant; damages organic compounds.
 - Hydroxyl radical ( $OH•$ ): Strong oxidant; damages all organic compounds.
- Enzymes for Oxygen detoxification
 - Catalase: $2H2O2 → 2H2O + O2$
 - Peroxidase: $H2O2 + NADH + H^+ → 2H_2O + NAD^+$
 - Superoxide dismutase: $O₂¯¯ + O₂¯ + 2 H^+→ H₂O2 + 0_2$

Growth Control

Most important factors:
- Nutrient
- Water
- pH
- Temperature
- Chemicals/antibiotics
  - Salt
  - Alcohol

Sterilization

Any process that eliminates/kills all forms of microbial life.
- Heat sterilization:
  - Autoclave: $121^oC$ , 15-21 psi, 20min/liter liquid
  - Flame sterilization: Only applicable to non-flammable solids.
  - Incineration: Applicable to solid wastes.
- Chemical sterilization: Plastic disposable items.
- Radiation sterilization.

Disinfectants and Antiseptics

Disinfectants
- Antimicrobial chemical agents (e.g., phenolic compounds, alcohols, synthetic detergents (quaternary ammonium compounds; QAC), gases (formaldehyde, ethylene oxide)).
- Kill microorganisms (but not all); endospores are the most resistant; some bacteria and viruses have tolerance.
- Applied to non-living objects (not on living tissue).
- Less effective than sterilization.
Antiseptics
- Similar to disinfectants but used on living tissues such as skin and throat mucosa (not for internal use, though).
- Some are true bactericidal while others are bacteriostatic.

Antibiotics

Differ from disinfectants and antiseptics; biosynthesized (produced by living organisms) and usually by-products of microbial metabolism (Bacillus, Penicillium, Streptomyces).
Can be administered internally (low toxicity to body cells while being toxic to bacterial invaders).
Antimicrobial/Antibacterial agents can also be chemically synthesized (chemotherapeutic agents).
Kirby-Bauer antibiotic testing (Disk diffusion antibiotic sensitivity testing)
- Evaluates susceptibility of a microorganism to an array of antibiotics (while all other variables held constant).
- Widely accepted standardized test (sanctioned by US FDA and *NCCLS).
- *NCCLS: the National Committee for Clinical Laboratory Standards